| Avian leukosis is caused by avian leukosis virus (ALV). ALV infection of chickens is widespread distributed and known to be capable of inducing a variety of tumor diseases and immunosuppression. As the ALVs have a capacity of vertical transmission, the population cleaning is the only effective way to prevent this disease by now. In the past10years, outbreaks of subgroup J avian leukosis has been widely recognized and largely reported in China. But little is known about other subgroups of ALV till now.In this study, by inoculation samples in DF-1cell culture, detection of p27antigen and PCR identification,7ALV strain were isolated from infected chickens of Chinese native breeds. Comparison of gp85gene nucleotide sequences between the isolates and other ALV strains of different subgroups indicated that,6A and19A had the highest homology with5reference strains of subgroup A (in the range of90.2%-99.4%);6B,7B,15B and16B had the highest homology with3reference strains of subgroup B (in the range of90.0%-99.0%);2J had the highest homology with4reference strains of subgroup J (in the range of92.9%-94.9%).In this study, the gp85gene of ALV-A and ALV-B was amplified by PCR, and cloned into expression vectors pET-32a(+) and pGEX-6P-1. Constructed recombinant plasmid were named pET-A-gp85, pET-B-gp85, pGEX-A-gp85and pGEX-B-gp85and transformed into E.coli BL21or BL21(DE3) respectively. Expression of recombinant fusion proteins were induced by IPTG and identified by SDS-PAGE.Recombinant fusion proteins His-A-gp85, GST-A-gp85and GST-B-gp85were purified with High-Affinity Ni-IDA Resin and High-Affinity GST Resin respectively. Six-week-old BALB/c mice were subcutaneously injected with purified GST-A-gp85or GST-B-gp85protein emulsified with Freund’s complete adjuvant, each one with200μg. Mice were subsequently injected two more times with purified GST-A-gp85or GST-B-gp85protein mixed with Freund’s incomplete adjuvant at two-week intervals, each one with200μg. An intraperitoneal booster of the same dose of GST-A-gp85and GST-B-gp85was given3days before splenocytes were collected and fused to SP2/0myeloma cells. The ELISA plates were coated with purified protein and hybridoma culture supernatants were screened by indirect ELISA method. Then selected the positive to subclone and obtained2hybridoma cell lines secreting monoclonal antibody anti-A-gp85antigen,named A5D1and A6C6; and2hybridoma cell lines secreting monoclonal antibody anti-B-gp85antigen, named B2C11and B6B7. The titers of ascetic fluid A5D1and A6C6were1:204800, B2C11and B6B7were1:409600. Subclass of monoclonal antibodies A5D1, B2C11and B6B7were IgG1, A6C6was IgG2b. Indirect immunofluorescence and Western-blot approved that the McAbs A5D1and A6C6were specific against purified protein GST-A-gp85, B2C11and B6B7were specific against purified protein GST-B-gp85. |
